Analysis of mutants that are affected in phosphate solubilisation activity

LUDUEñA, L., ANGELINI, J., FABRA, A., TAURIAN, T.

Congreso; VII Congreso Argentino de Microbiología General "SAMIGE del bicentenario"; 2011

Phosphorus, despite its abundance in nature, is a limiting factor for plant growth. The low levels are due to the fact that soluble soil phosphorus reacts with ions that cause the precipitation or fixation, reducing their availability to plants. Many soil microorganisms are involved in processes that affect the transformation of phosphorus from soil and therefore are an integral part of its cycle. The main mechanism by which microbial phosphate compounds are mobilized is the release of organic acids of low molecular weight with a significant decrease of pH values. It has been described a synergistic effect between the production of exopolysaccharides (EPS) and organic acids on the solubilization of inorganic phosphates. Understanding the basic mechanisms related to the solubilization of these compounds will allow the application of microorganisms with an effective and high capacity to increase the available levels of phosphorus for plants. Objective: To identify genes involved in phosphate solubilising mechanism. Methods: Getting transpositional insertion mutants: The plasmid pUTminiTn5Km was transferred to the strain Serratia sp S119 by triparental conjugation using Escherichia coli CC118 ëpir and E. coli DH5á as donor and helper strains, respectively (Simon et al., 1983). Selection of mutants: in NBRIP-BPB (Mehta and Nautiyal, 2001) containing tricalcium phosphate supplemented with appropriate antibiotics and determination of phosphate solubilisation halo in NBRIP-BPB.  Isolation of genomic DNA (Walsh et al.,1991). ERIC-fingerprints (Versalovic et al., 1989). MiniTn5 detection by PCR. Viability of mutants by growth curves in NBRIP broth determining CFU/ml. Quantification of soluble phosphorus released (Fiske and Subbarow, 1925) in NBRIP containing glucose or glycerol as carbon source and analysis of pH from 2 to 48 hs of growth. Qualitative determination of EPS production in King B medium supplemented with calcofluor (0.002%). Data were analyzed using test "t" of Student and considering a significant difference at p <0.05. Results: There were selected 3 colonies (4A, 5A and 6A) that showed a significant reduction in their phosphate solubilization halo on NBRIP plates. These strains showed identical ERIC profiles being thus isogenic mutants of Serratia sp S119. In all of them it was possible to confirm the presence of the insert. Viability of mutants 4A and 5A was similar to that of wild type while mutant 6A showed a significant decrease in CFU/ml. The mutant 4A released significantly less amounts of soluble phosphorus compared to the wild type after 8 and 10 hs of growth, while in the mutant 5A this diminution was only observed after 8 hs of growth. The quantity of soluble phosphorus released was negatively correlated with pH values ​​of the medium for all strains. The presence of a non-fermentative carbon source such as glycerol